Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis

Abstract

Introduction: The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-gamma (IFN-gamma) in these processes.

Methods: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-gammaR KO MSC.

Results: Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-gammaR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.

Conclusions: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

Figure 1

Phenotype and differentiation potential of mesenchymal stem cells (MSCs). Bone marrow cells of DBA/1 wild-type and interferon-gamma receptor knockout (IFN-γR KO) mice were cultured in Murine Mesencult medium and phenotyped. (a-d) MSCs were incubated with the indicated antibodies and analyzed by flow cytometry. Grey histograms show stained cells, and black lines represent cells incubated with isotype controls. Wild-type MSCs were analyzed at passages 3, 4, and 7 (a) and IFN-γR KO MSCs were analyzed at passages 2 and 12 (b) for expression of CD11b, CD45, and Sca-1. Likewise, other phenotypic markers were analyzed on wild-type (c) and IFN-γR KO (d) MSCs. (e) MSCs were cultured to confluency in Murine Mesencult medium and then transferred to adipogenic or osteogenic differentiation medium for 21 days, followed by Oil Red O or Alizarin Red staining, respectively (original magnification × 10). The inset in the lower right panel represents an enlargement of the adipocyte indicated by the arrow.

Figure 2

Mesenchymal stem cells (MSCs) inhibit the anti-CD3-induced proliferation of CD4⁺ T cells in vitro. (a) CD4⁺ T cells (5 × 10⁴ cells) and accessory cells (5 × 10⁴ cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or interferon-gamma receptor knockout (IFN-γR KO) MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [³H]TdR. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of four cultures ± standard error of the mean (SEM). Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test). (b) Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4⁺ T cells (5 × 10⁴ cells) and accessory cells (5 × 10⁴ cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated numbers of mitomycin c-treated wild-type or IFN-γR KO MSCs for 72 hours. The proliferation of CD4⁺ T cells was analyzed by detection of CFSE dilution by flow cytometry. The percentage inhibition (100 × [(percentage of proliferating CD4⁺ cells not treated with MSCs -- percentage of proliferating CD4⁺ cells treated with MSCs)/percentage of proliferating CD4⁺ cells not treated with MSCs]) by increasing numbers of MSCs is shown. Each result represents the mean of three cultures ± SEM. Results are representative of two independent experiments. * P < 0.05 for comparison with wild-type MSCs (Mann-Whitney U test).

Figure 3

Mesenchymal stem cells (MSCs) inhibit the proliferation of CD4⁺ T cells in vitro by induction of nitric oxide and prostaglandin E₂ (PGE₂). (a) CD4⁺ T cells, in the presence of accessory cells, were stimulated with 3 μg/ml anti-CD3 for 48 hours. Interleukin-17 (IL-17) and interferon-gamma (IFN-γ) levels in the supernatant of these cultures were analyzed by Bio-Plex protein array system. Bars represent averages of three values ± standard error of the mean. (b-d) Wild-type MSCs were stimulated with IL-17 (20 ng/mL) or IFN-γ (100 U/mL) or both for 48 hours. cDNA samples were prepared and subjected to quantitative polymerase chain reaction analysis. The relative quantity of target mRNA levels was normalized for 18S RNA. Relative levels of programmed death ligand-1 (PD-L1) (b), inducible nitric oxide (iNOS) (c), and cyclo-oxigenase-2 (COX-2) (d) are shown. Bars represent averages of two values. ND, not detectable. (e) CD4⁺ T cells (5 × 10⁴ cells) and accessory cells (5 × 10⁴ cells) were incubated with 3 μg/ml anti-CD3 antibody and the indicated number of mitomycin c-treated wild-type MSCs for 72 hours and pulsed for the last 16 hours with 1 μCi of [³H]TdR. Co-cultures were grown in the absence (control) or presence of 200 μM 1-methyl-DL-tryptophan, 10 μM indomethacin, or 10 μM GW274150. The percentage inhibition (100 × [(radioactivity in cultures without MSCs -- radioactivity in cultures with MSCs)/radioactivity in cultures without MSCs]) by increasing numbers of MSCs is shown.

Figure 4

Treatment with mesenchymal stem cells (MSCs) of wild-type or interferon-gamma receptor knockout (IFN-γR KO) origin does not influence the development of collagen-induced arthritis in DBA/1 mice. Mice were immunized on day 0 with collagen type II (CII) in complete Freund's adjuvant and injected intravenously with MSCs on day 16 and day 23. The mean arthritic score (a) and the cumulative incidence of arthritis (b) in DBA/1 mice treated with phosphate-buffered saline (PBS), wild-type MSCs, or IFN-γR KO MSCs are shown. Error bars represent standard error of the mean (SEM). (c) On day 46, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (d) Forty-two days after immunization, five mice in each group were challenged with 10 μg of CII in the right ear and vehicle in the left ear. Delayed-type hypersensitivity responses were measured as the percentage of swelling (100 × [(thickness of the right ear -- thickness of the left ear)/thickness of the left ear]) at the indicated times. Histograms indicate averages ± SEM. (e) On day 19 after immunization with CII in complete Freund's adjuvant, DBA/1 wild-type mice were injected intravenously with 1 × 10⁶ wild-type MSCs, IFN-γR KO MSCs, or PBS, followed by an administration of 10 μg of anti-CD3 antibody on day 20. On day 21, in vivo T-cell proliferation was measured by detection of 5-ethynyl-2' -deoxyuridine (EdU) in the T-cell populations in the spleen and lymph nodes by fluorescence-activated cell sorting analysis. The percentages of EdU-positive cells in the CD4⁺ and CD8⁺ populations in the spleen and lymph nodes are shown. Histograms represent averages of four mice ± SEM.